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Biomedical subjects

F E Regnier

Publications and source records attributed to F E Regnier.

At least 19 recordsLinked to original sources
PubMed1992

Ultramicro enzyme assays in a capillary electrophoretic system.

This paper describes an ultramicro method for achieving enzyme assays. Enzyme saturating concentrations of substrate, coenzyme when appropriate, and running buffer were mixed and used to fill a deactivated fused-silica capillary in a capillary zone electrophoresis apparatus. The enzyme glucose-6-phosphate dehydrogenase was injected by either electrophoresis or siphoning and mixed with the reagents in the capillary by electrophoretic mixing. Enzyme activity was assayed by electrophoresing the product, reduced nicotinamide adenine dinucleotide phosphate, to the detector where it was detected at 340 nm. Under constant potential, the transport velocity of enzyme and the product was generally different. This caused product to be separated from the enzyme after it was formed. Because product formation was much faster than the rate of enzyme-product separation, product accumulated. The amount of accumulated product was inversely related to operating potential. In the extreme case, the operating potential was zero. Zero potential assays were generally carried out by electrophoresing the enzyme partially through the capillary and then switching to zero potential. This capillary was left at zero potential for several minutes to allow additional product to accumulate. After this additional amplification step, potential was again applied and the product transported to the detector. Product formed under constant potential appears as a broad peak with a flat plateau. When the voltage is switched to zero at intermediate migration distance, a peak will be observed on top of this plateau. Either the eight of the plateau or the area of the peak may be used to determine enzyme concentration. The lower limit of detection was 4.6.10(-17) mol of glucose-6-phosphate dehydrogenase.

Electrophoresis↗
PubMed1992

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.

Acrylic Resins↗
PubMed1992

Kinetic chromatographic sequential addition immunoassays using protein A affinity chromatography.

A new type of chromatographic immunoassay based on sequential addition is described. On a protein A column, the antibody, the sample containing the antigen, and then a known amount of antigen are sequentially injected. This assay is designed to shorten analysis times and reduce complexity of dual-column chromatographic immunoassays, circumvent desorption buffer interferences common to affinity chromatography, and eliminate the need for tagged molecules. This new technique is named kinetic immunochromatography sequential addition (KICQA). Because of its kinetic nature, flow rate will have a large effect on KICQA, and the impact of changing flow rate is studied extensively. By use of various amounts of antibody, the dynamic range of KICQA is shown to be selectable over 2.5 orders of magnitude. Finally, KICQA was used to determine transferrin and albumin in human serum. Both analytes show good agreement with their respective reference methods, and an albumin assay was performed in under 1 min.

Antigen-Antibody Complex↗
PubMed1992

Automated real-time immunoassay of biomolecules.

ImmunoDetection is a novel technique combining perfusion chromatography technology with antibodies to perform the steps of an immunoassay in a flow-through column format. Sensitive and precise measurements are performed in seconds to minutes using automated liquid chromatography instrumentation.

Antibodies↗
PubMed1992

Contribution of soluble aluminium species to absorption of aluminium from the rat gut in situ.

1. Rat gut perfusion studies in vivo at pH 4, 6 or 8 using aluminium chloride or equimolar aluminium chloride and sodium citrate showed that elevated plasma aluminium concentrations were associated with aluminium solubility in the perfusion. Elevated plasma aluminium levels and soluble aluminium in the perfusate occurred with perfusion of equimolar aluminium chloride and sodium citrate at all three pH values. 2. Partitioning studies in vitro, utilizing water and ethyl acetate, revealed that uncomplexed aluminium exhibited maximum partitioning into the ethyl acetate phase at pH 2.5. When complexed with citrate, aluminium exhibited partitioning over a much broader pH range, pH 2.5-8.0. 3. A direct linear relationship was observed between the soluble aluminium concentration of the perfusate and the increase in the plasma aluminium level, suggesting that soluble aluminium is absorbed by a passive diffusion mechanism.

Aluminum↗
PubMed1991

Process monitoring by parallel column gradient elution chromatography.

A system was developed that generates two gradients simultaneously, 180 degrees out of phase. Essential elements of the system were a pump delivering solvent A at a constant velocity, a pump delivering solvent B at a constant velocity, two mixing chambers, and a ten-port valve. By use of this gradient device and two reversed-phase columns packed with 1000-A pore diameter, 8-microns particle size macroporous poly-(styrene-divinylbenzene) resin, six proteins were separated in 40 s in a single gradient cycle.

Chromatography↗
PubMed1991

Capillary electrophoretic separations of proteins using nonionic surfactant coatings.

Capillary zone electrophoretic separations of proteins have been achieved by using nonionic surfactant coated capillaries. Capillaries were prepared by derivatization of the silica surface with octadecylsilane followed by the deposition of a layer of nonionic surfactant from an aqueous solution above the critical micelle concentration. This coating is of sufficient thickness and hydrophilicity to reduce both protein adsorption and electroosmotic pumping. This hydrophilic coating reduces electroosmotic pumping 5-8-fold while resolving proteins quickly and efficiently with good recovery. The coating provides a stable and reproducible means of deactivation, while the rate of electroosmotic pumping stays relatively constant throughout the pH range 4-11. This allows the pH to be varied to enhance selectivity without adversely affecting the flow rate.

Electrophoresis↗
PubMed1991

Semipermeable-surface reversed-phase media for high-performance liquid chromatography.

Polyoxyethylene was both adsorbed hydrophobically (through the use of non-ionic surfactants) and covalently bonded to reversed-phase high-performance liquid chromatographic packings, thereby establishing a semipermeable hydrophilic layer over the alkylsilane surface. This layer restricts proteins from adsorbing to the alkylsilane phase while permitting penetration and chromatographic separation of small molecules. Biological fluids containing low-molecular-weight analytes may be injected directly, without sample pretreatment or the use of micellar eluents. In the case of adsorbed coatings, surfactant loading was determined primarily by the surface area (over the reversed phase) occupied by the polyoxyethylene head group. Semipermeability of the hydrophilic layer was demonstrated by observing changes in retention of both small molecules and proteins with increasing eluent ionic strength. Coated column stability was evaluated with regard to cumulative eluent volume and repeated serum injections.

Adsorption↗
PubMed1991

Coated hydrophilic polystyrene-based packing materials.

A very hydrophilic high-performance liquid chromatographic base support was created from microparticulate, macroporous poly(styrene-divinylbenzene) beads. An organic monomer containing cross-linking functionalities was coated on the poly(styrene-divinylbenzene), followed by a catalyzed cross-linking reaction. The coatings formed contain only stable chemical bonds (e.g., C-C, C-O-C), and easily-derivatized hydroxyl moieties. This coated base support was evaluated for hydrophilicity, chemical stability, solvent compatibility, rigidity, and irreversible adsorption. Derivatives of the coated base support were made and applied in various modes of chromatography.

Cations↗
PubMed1991

Perfusion chromatography.

Perfusion chromatography is a technique based on fluid dynamics for reducing stagnant mobile phase mass transfer in liquid chromatography. This is achieved by using supports with large pores that allow mobile phase to flow through particles.

Chromatography↗
PubMed1991

Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection.

The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves are established by using a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The precision is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is greater than or equal to 96%. Sample analysis times are on the order of 20 min. The HPAC technique gives results in absolute units of concentration that correlate well with binding capacity values determined by radioimmunoassay.

Antibodies↗
PubMed1990

Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography.

This paper reports a new technique for reducing resistance to stagnant mobile phase mass transfer without sacrificing high adsorbent capacity or necessitating extremely high pressure operation. The technique involves the flow of liquid through a porous chromatographic particle, and has thus been termed "perfusion chromatography". This is accomplished with 6000-8000 A pores which transect the particle. Data from electron microscopy, column efficiency, frontal analysis and theoretical modelling all suggest that mobile phase will flow through these large pores. In this manner, solutes enter the interior of the particles through a combination of convective and diffusional transport, with convection dominating for Peclet numbers greater than one. The implications of flow through particles on bandspreading, resolution and dynamic loading capacity are examined. It is shown that the rate of solute transport is strongly coupled to mobile phase velocity such that bandspreading, resolution of proteins and dynamic loading capacity are unaffected by increases in mobile phase velocity up to several thousand centimeters per hour. The surface area of this very large-pore diameter material is enhanced by using a network of smaller, 500-1500 A interconnecting pores between the throughpores. Scanning electron micrographs show that the pore network is continuous and that no point in the matrix is more than 5000-10,000 A from a through-pore. As a consequence, diffusional path lengths are minimized and the large porous particles take on the transport characteristics of much smaller particles but with a fraction of the pressure drop. Capacity and resolution studies show that these materials bind and separate an amount of protein equivalent to that of conventional high-performance liquid chromatography as well as low performance agarose-based media at greater than 10-100 times higher mobile phase velocity with no loss in resolution.

Chromatography, High Pressure Liquid↗
PubMed1990

Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis.

A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length.

Animals↗
PubMed1990

Microenvironmental contributions to the chromatographic behavior of subtilisin in hydrophobic-interaction and reversed-phase chromatography.

Genetically engineered variants were used to examine how microenvironmental changes in the S1 substrate binding subsite of subtilisin contribute to chromatographic behavior of proteins on hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC) columns. Gradient elution studies over a wide pH range showed that conditions could be found where a HIC support could separate proteins varying by one amino acid. Although all single-site variants could not be separated by HIC, this chromatographic mode was found to be complementary to cation-exchange chromatography for the separation of such variants. RPC was found to be of much less utility in the resolution of variant proteins. Retention and resolution of subtilisin variants was found to vary on RPC with the concentration and type of mobile phase pairing agent.

Amino Acids↗
PubMed1990

Thermodynamic model for electrostatic-interaction chromatography of proteins.

A thermodynamic model derived by Record et al. [M. T. Record, Jr., Biopolymers, 14 (1975) 2137 and M. T. Record, Jr., C. F. Anderson and T. M. Lohman, Q. Rev. Biophys., 11 (1978) 103] from Wyman's linkage theory [J. Wyman, Adv. Protein Chem., 19 (1964) 223] using Manning's condensation model [J. Manning, J. Chem. Phys., 51 (1969) 924] was extended to electrostatic interaction chromatography. Mixed, electrostatic and hydrophobic interactions of a model protein, ovalbumin were characterized by ion and water release.

Chromatography↗
PubMed1989

Single amino acid contributions to protein retention in cation-exchange chromatography: resolution of genetically engineered subtilisin variants.

Genetically engineered proteins were used to determine the amino acid contributions of surface residues to subtilisin retention in cation-exchange chromatography. Crystallographic data were used to correlate the observed chromatographic behavior with enzymatic structure. Retention times of variants in gradient elution varied by as much as 33% compared to the wild type. The role of both charged and uncharged residues was investigated in isocratic separations and found to significantly influence protein retention in this electrostatically dominant separation method. This study demonstrates the ability of ion-exchange chromatography to discriminate between protein variants differing by a single residue in 275 amino acids.

Amino Acid Sequence↗
PubMed1989

Dual-column immunoassays using protein G affinity chromatography.

Tandem protein G affinity and reversed-phase chromatography (RPC) columns, coupled with a switching valve, were used for on-line immunoassays of antibodies and antigens. Columns with reversibly immobilized antibodies were prepared by adsorbing antibodies on the protein G column. Following antigen capture in the immunoaffinity column, antigen-antibody complexes were desorbed, dissociated, and transferred to the RPC column where they were separated and quantified. This system was used to determine the titer of a rabbit anti-human transferrin antibody sample with a precision of +/- 2%. Quantitation of human transferrin in human serum had a precision of +/- 6% and showed good agreement with rate nephelometry. The linear dynamic range for the transferrin, antigen immunoassay was 5 x 10(1) to 1 x 10(5) ng with a precision of +/- 3.5%.

Chromatography, Affinity↗